The invention relates to peptide analogues from prostate acidic phosphatase (PAP) and their use as vaccines. In particular, the analogue includes the change of alanine to leucine at position 115 of PAP which has been found to be particular advantageous.
Prostate cancer is the most common form of male cancer in developed countries and cases are rising among men under 50. Every year, more than 32,000 British men are diagnosed and 10,000 die from the disease [1,2]. At present there is no standard treatment available for patients with biochemical recurrence in the absence of radiographically-visible metastases [3]. A long-held dream of tumour immunologists is to harness the specificity and sensitivity of the immune response and induce specific protective anti-tumour immunity. Unfortunately, for most cancers, the specific antigenic determinants on which vaccination strategies can be based are weakly immunogenic self-antigens which are comparatively poor at inducing robust, protective anti-tumour responses. Developing cancer vaccines that can overcome immune evasion and the tolerogenic capacity of self-antigens and induce protective immune responses is therefore essential for the development of new immunotherapies for aggressive disease.
Several tumour-associated antigens have been reported to be expressed by prostate cancer cells. These include prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), and prostate acidic phosphatase (PAP) [4]. The prostate-restricted expression and overexpression of PAP in prostate cancer makes this antigen an ideal candidate on which to base a cancer vaccine [5]. The potential of the PAP antigen has recently been illustrated in the form of an FDA-approved vaccine for advanced prostate cancer which has been demonstrated to increase the overall survival of patients. The vaccine consists of in vitro patient-derived mononuclear cells transfected with the entire prostatic acid phosphatase (PAP)-protein fused to GM-CSF (Provenge, sipuleucel-T) [6]. However, the production of an entire protein vaccine is time-consuming and costly, and the exact region of the PAP protein which is responsible for eliciting the therapeutic effect remains unknown. Furthermore, in other settings, vaccination using long peptides rather than whole protein has been shown to provide a more efficient and robust protective immune response [7]. An important feature of PAP in the context of a vaccine for prostate cancer is that sequences contained within the PAP molecule exhibit a high degree of homology between the murine and human proteins.
This means that pre-clinical (murine) models focussed on inducing protective anti-tumour immunity using a PAP-based vaccine present similar challenges relating to the presence of tolerance to ‘self’ antigens such as PAP to those that are encountered in the clinical setting. Results obtained using such a mouse model therefore have a better potential for being successfully translated into patients.
WO 2005/090560 relates to peptides from PAP as positions 135 and 161 which were identified as candidate peptides for vaccine-based immunotherapy. The PAP 135 sequence ILLWQPIPV (amino acids 33-41 of SEQ ID NO: 1) showed a strong binding to HLA-A2.
The current invention focuses on a mutation at position 115 of PAP from Ala to Leu. This has been unexpectedly found to produce enhanced immune response. This suggests that PAP peptides encoding the analogue, or DNA vaccines encoding the analogue will produce an improved immune response compared to sequences with the native PAP 115 sequence. The analogue has also been found to work as both small and large peptides, allowing the production of peptides with multiple epitopes.
This was unexpected. Changing the amino acid at position 115 to, for example isoleucine, valine or methionine did not enhance the HLA binding score and immune response.
The invention provides a polypeptide comprising the sequence SLMTNLAAL (SEQ ID NO: 8) having HLAA2 haplotype binding activity or a polynucleotide encoding said polypeptide.
That is, this is the polypeptide comprising the sequence from Ser 13 to Leu 21 of the amino acid sequence shown in FIG. 1 or SEQ ID No: 1.
Polypeptides may comprise at least 9, 15, 20, 25, 30, 35, 45 or 55 or less than 60 amino acids of the native PAP amino acid sequence, provided that the sequence contains the polypeptide sequence corresponding to Ser 13 to Leu 21.
Polypeptides or polynucleotides comprising the sequence Met 12 to Gly 26 of the amino acid sequence shown in FIG. 1 or SEQ ID No: 1 are also provided. That is, they comprise the sequence MSLMTNLAALFPPEG (SEQ ID NO: 9). These are being found to have a Syfpeithi score of 33, and have HLA-DR1 haplotype binding activity, as well as HLA-A2 activity.
The Syfpeithi database is a database of more than 7000 peptide sequences known to bind class I and class II MHC molecules compiled from published records. This allows potential motifs and their ability to bind HLA haplotypes to be identified with a high degree of accuracy. The HLA haplotype binding activity may be determined using that database (www-dot-syfpeithi-dot-de).
A further embodiment of the invention provides a polypeptide or polynucleotide comprising the sequence Asp 8 to Phe 22 of the amino acid sequence shown in FIG. 1 or SEQ ID No: 1. That is, it comprises the sequence DRTLMSLMTNLAALF (SEQ ID NO: 10). This is a 15 Mer. The mutation to use leucine has been found to increase the Syfpeithi score from 22 to 30. It has the following haplotype activity, HLA-DR4 and HLA-A2 activity.
A still further aspect of the invention provides a polypeptide comprising the sequence Ser 13 to His 42 of the amino acids shown in FIG. 1 or SEQ ID No: 1.
The sequence includes not only the 9 Mer sequence SLMTNLAAL (SEQ ID NO: 8) but additionally comprises the sequence ILLWQPIPV (amino acids 33-41 of SEQ ID NO: 1) which has HLA-A2 activity.
A still further aspect of the invention provides a polypeptide or polynucleotide according to the invention comprising the sequence Arg 9 to Pro 23 of the amino acid sequence shown in FIG. 1 or SEQ ID No: 1. That is, RTLMSLMTNLAALFP (SEQ ID No: 14). This has been found to increase the Syfpeithi score from 22 to 23 with the leucine mutation. It has the following HLA-haplotype: HLA-DR1 and HLA-A2 activity.
A still further aspect of the invention provides the sequence Tyr 1 to His 42 of the amino acid sequence shown in FIG. 1 or SEQ ID No: 1. The advantage of this sequence is that is contains a number of different haplotype sequences.
FIG. 1 shows the same polypeptide sequence, but with different leucine codons at amino acid position 14. That is, the leucine codon may be selected from CTT, CTC, CTA, CTG, TTA or TTG. Typically the leucine codon is CTT.
That is, the native sequence for PAP is mutated at position 115 so that instead of the sequence encoding alanine, it then encodes leucine.
The polypeptide may be incorporated into a polypeptide sequence encoding at least a part of an antibody or immunoglobulin, for example through the use of the commercially available technology known as “Immunobody™” described below. The polypeptide may also be fused, for example, to a part of a gene sequence from granulocyte-macrophage colony stimulating factor (GM-CSF), such as that provided by the GVAX technology described below.
The polypeptide may also be fused to a sequence, or used in conjunction with an adjuvant, comprising a sequence from HSP-70, Shiga toxin, alpha-GAL-Ser, or TLR agonists such as CpG or PolyIC.
HSP-70 has been known to be used as an adjuvant in combination with DNA vaccines. This heat shock protein induces antigen-specific cellular and humoral immunity (see for example Zang X et al, J Gene Med. 2007 9(8) 715-26).
Microbial proteins in the cytosol of host cells activate CD8+ cytotoxic T lymphocytes (CTLs). Once activated, CTLs lyse infected cells and secrete cytokines that stimulate other immune cells at the site of infection. Because of this modified bacterial toxins have been used to deliver vaccine antigens. Accordingly, the polypeptides of the invention may be fused to or be used in combination with a bacterial toxin such as Shiga toxin, anthrax toxin or diphtheria toxin.
Synthetic oligodeoxynucleotides containing unmethylated CpG motifs, have been found to trigger cells that express TOLL-like receptor 9, to mount immune responses characterised by the production of Th1 and proinflammatory cytokines. When used as vaccine adjuvants CpG improves the function of professional antigen-presenting cells and boosts the generation of humoral anti-cellular vaccine-specific immune responses. See for example, Bode C et al, Expert Rev Vaccines 2011 10(4) 499-511.
PolyIC is a synthetic double-stranded RNA made of polyinosine-polycytidylic acid that can activate the immune response. It is commercially available from, for example, InvivoGen, under the trademark VacciGrade, and acts as a vaccine adjuvant.
Alpha-GAL-Ser (Alpha-Galactosylceramide) is a glyco lipid composed of alpha-linked sugar and lipid moieties. It has been described for use as a vaccine adjuvant (see, for example, Li X, Fujio M, Imamura M, Wu D, Vasan S, Wong C H, Ho D D, Tsuji M. Proc Natl Acad Sci USA. 2010 Jul. 20; 107(29):13010-5. doi: 10.1073/pnas.1006662107
Polypeptides may be fused to a ubiquitin or secretory leader sequence. This allows a polypeptide produced by a polynucleotide encoding the protein to be secreted from the cell.
pVAX1 or GVAX vectors comprise a polynucleotide of the invention.
pVAX1™ is a 3.0 kb plasmid vector designed for use in the development of DNA vaccines. It is supplied by Invitrogen a part of Life Technologies Corporation and is supplied as catalog number V160-20 (as of 2 Mar. 2012). The sequence of pVAX1 is shown in the sequence listing SEQ ID No: 12. A schematic diagram of pVAX1 is shown in FIG. 2. The vector is also available and may be used with a lacZ gene, containing β-galactosidase as a control vector. The kanamycin resistance gene of pVAX1 may be replaced by a non-antibiotic based marker.
GVAX is a granulocyte-macrophage colony stimulating factor (GM-CSF) gene transfected tumour cell vaccine which has been demonstrated to produce good vaccine activity with other PAP peptides, as have other DNA or polynucleotide vaccines (Nemunaitis J., Expert Rev. Vaccines 2005 4(3) 259-74), McNeel D G et al J. Clin. Oncol. 2009, 27(25) 4047-4054, Geary S M et al. Oncoimmunology 2013, 2(5) 24523).
An alternative DNA vaccine may utilise Immunobody™ DNA vaccine technology. Immunobody™ is a human antibody or fusion protein which is engineered to express helper cell and CTL epitopes from tumour antigens that are overexpressed by cancer cells. The Immunobody™ technology is commercially available from Scancell Ltd, Nottingham U.K. See, for example, Pudney et al Eur. J. Immunol. 2010, 40:899-910 and Durrant L G et al Expert. Rev, Vaccines 2011 10(7) 1088. The technology inserts tumour associated epitopes into the structure of antibodies. Antibodies have been found to be good DNA vectors for stimulating immune responses. Responses are 100-1000 fold more effective than protein, peptide or antigen DNA immunisation.
Polynucleotides encoding the peptide, alone or fused to a polynucleotide vaccine as described above, may be administered, for example, intravenously or intramuscularly. Immunobody™-based vaccines have been previously found to persist at intramuscular injection sites for 90 days and lymph nodes for 7 days and induce strong CD8+ responses (Durrant et al Supra).
The polynucleotide sequence encoding the peptides of the invention may also be modified by attaching to the sequence encoding the peptide, a ubiquitin sequence and/or a leader sequence for allowing the secretion of the peptide, prior to incorporation into a vector such as those described above.
Vaccines comprising the polynucleotides or polypeptides of the invention are also provided. Polypeptides or polynucleotides of the invention may be used as part of a vaccine against prostate cancer.
Methods of preventing or treating prostate cancer comprise administering a polypeptide or polynucleotide of the invention are also provided.